Continuous flow methods for monitoring time dependent reactions

ABSTRACT

Methods for monitoring time dependent reactions that comprise providing a flow channel, typically microscale in dimension, flowing at least two reagents into the flow channel and varying the flow rate of the mixture through the flow channel. By increasing and/or decreasing the flow rate of the reagent mixture from the point of mixing to the point of detection, one alters the amount of reaction time, allowing monitoring reaction kinetics over time.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/774,531, filed Jan. 31, 2001, which issued as U.S. Pat. No. 6,589,729on Jul. 8, 2003, which claims priority to Provisional U.S. PatentApplication No. 60/180,235, filed Feb. 4, 2000; Ser. No. 60/182,086,filed Feb. 11, 2000; and Ser. No. 60/211,827, filed Jun. 15, 2000, eachof which is hereby incorporated herein by reference in its entirety forall purposes.

BACKGROUND OF THE INVENTION

The biological and chemical sciences, much like the electronicsindustry, have sought to gain advantages of cost, speed and conveniencethrough miniaturization. The field of microfluidics has gainedsubstantial attention as a potential solution to the problems ofminiaturization in these areas, where fluid handling capabilities areoften the main barrier to substantial miniaturization.

For example, U.S. Pat. Nos. 5,304,487, 5,498,392, 5,635,358, 5,637,469and 5,726,026, all describe devices that include mesoscale flow systemsfor carrying out a large number of different types of chemical, andbiochemical reactions and analyses.

Published international patent application No. WO 96/04547 to Ramseydescribes microfluidic devices that incorporate electrokinetic means formoving fluids or other materials through interconnected microscalechannel networks. Such systems utilize electric fields applied along thelength of the various channels, typically via electrodes placed at thetermini of the channels, to controllably move materials through thechannels by one or both of electroosmosis and electrophoresis. Bymodulating the electric fields in intersecting channels, one caneffectively control the flow of material at intersections. This createsa combination pumping/valving system that requires no moving parts tofunction. The solid state nature of this material transport systemallows for simplicity of fabricating microfluidic devices, as well assimplified and more accurate control of fluid flow.

Published international patent application No. WO 98/00231 describes theuse of microfluidic systems in performing high throughput screening oflarge libraries of test compounds, e.g., pharmaceutical candidates,diagnostic samples, and the like. By performing these analysesmicrofluidically, one gains substantial advantages of throughput,reagent consumption, and automatability.

Despite the above-described advances in the field of microfluidics,there still exist a number of areas where this technology could beimproved. For example, while electrokinetic material transport systemsprovide myriad benefits in the microscale movement, mixing andaliquoting of fluids, the application of electric fields can havedetrimental effects in some instances. For example, in the case ofcharged reagents, electric fields can cause electrophoretic biasing ofmaterial volumes, e.g., highly charged materials moving at the front orback of a fluid volume. Solutions to these problems have been previouslydescribed, see, e.g., U.S. Pat. No. 5,779,868. Alternatively, where oneis desirous of transporting cellular material, elevated electric fieldscan, in some cases, result in a perforation or electroporation, of thecells, which may affect their ultimate use in the system.

In addition to these difficulties of electrokinetic systems,microfluidic systems, as a whole, have largely been developed asrelatively complex systems, requiring either complex electrical controlsystems or complex pump and valve systems, for accurately directingmaterial into desired locations. Accordingly, it would be generallydesirable to provide microfluidic systems that utilize simplifiedtransport systems, but that are also useful for carrying out importantchemical and/or biochemical reactions and other analyses. The presentinvention meets these and a variety of other needs.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides for a method ofmonitoring a time-dependent reaction. The method comprises introducingfirst and second reagents into a first flow channel wherein the reactionis initiated with respect to at least a first reagent, to form a firstreaction mixture. The first mixture is transported along the flowchannel, past a detection zone which detects an extent of the reaction.The flow rate of the first mixture is varied along the flow channel tovary an amount of time between mixing of the first and second componentsand detection of the extent of the reaction at the detection zone. Theresult of an interaction is then monitored between the first and secondreagents.

Another aspect of the present invention is a system for monitoring atime dependent reaction. The system comprises a body containing at leasta first flow channel. The first flow channel is fluidly connected to asource of a first reagent and a source of a second reagent. A flowcontroller is operably coupled to the flow channel, which containsprogramming to provide a varying flow rate of a fluid into and throughthe flow channel.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A–1C schematically illustrate the principles of flow ratevariation as it relates to reaction parameters, in accordance with thepresent invention; FIG. 1A shows two reagents being brought together ina flow channel, e.g., from two flow channels connected to a main flowchannel; FIG. 1B illustrates a situation where the flow rate isincreased through the main flow channel of FIG. 1A; FIG. 1C illustratesthe situation where the flow rate is decreased through the main flowchannel of FIG. 1A.

FIG. 2 schematically illustrates the layered construction of a simplemicrofluidic device.

FIG. 3 schematically illustrates the channel layout for a microfluidicdevice that is particularly useful in practicing the methods of thepresent invention.

FIG. 4 is a plot of the response kinetics of the P2u receptor to UTP, asmeasured using the methods and systems of the invention.

FIG. 5 is a plot of the dose response of the P2u receptor to UTP, fromthe response kinetics shown in FIG. 4.

FIG. 6 is a plot of the response kinetics of the M1 muscarinic receptorto Carbachol, as measured using the methods and systems of theinvention.

FIG. 7 is a plot of the dose response of the M1 muscarinic receptor toCarbachol, as determined from the response kinetics shown in FIG. 6.

FIG. 8 illustrates an alternative system for monitoring time dependentreactions.

DETAILED DESCRIPTION OF THE INVENTION

I. General Description

The present invention generally provides methods for monitoring timedependent reactions. Monitoring of time-dependent reactions isparticularly useful in the analysis of reaction kinetics in chemical,biochemical and biological interactions, in order to ascertain themechanics of those reactions and further elucidate affectors of thosereactions.

Generally, the methods of the present invention typically comprise flowthrough assay methods where the reactants, e.g., first and secondreagents, are flowed concurrently into and through a flow channel,whereupon the reagents mix in a reaction mixture. The reagents interactand react to produce products of the particular reaction. The amount ofproduct produced in a given reaction is a measure of the extent of thereaction that is being analyzed. The reaction mixture is flowed past adetection zone at which the accumulated products (or depleted reactants)of the reaction are detected. The reaction time is the length of timebetween the initiation of the reaction of interest, e.g., the mixing ofreactive reagents, and the detection time.

Although generally described herein as a reaction between two reagents,it will be appreciated that the methods described herein are equallyapplicable to reactions carried out on a single reagent, e.g., reactionsthat are initiated by means other than combination with another reagent.Such method of initiating reactions include, e.g., photoactivatedreactions, where a particular reagent is rendered more reactive byvirtue of exposure to light, as well as thermally activated reactionsthat are activated by heat. Similarly, the response of the reagent(s) toexternal stimuli such as light and/or heat can be measured as thereaction of interest, e.g., photo or thermal degradation of reagents,etc.

When the flow rate of the flowing mixture through the flow channel ismaintained constant, the extent of the reaction detected at thedetection zone will remain constant, barring any other intervention inthe reaction. In accordance with the present invention, however, a timecourse for the reaction is obtained by varying the flow rate of thereaction mixture through the flow channel and past the detection point,e.g., either slowing it down or speeding it up, thus giving the reactionmixture a longer or shorter time frame in which to react. FIGS. 1A, 1Band 1C schematically illustrate the effect of flow rate variations(indicated by arrows of differing length within the main channel) on theextent of a reaction that is detected in the systems described herein.For example, in FIG. 1A, two reagents are shown being brought togetherin the flow channel, e.g., from two flow channels connected to a mainflow channel. At a given flow rate, the amount of time “t”, e.g., inseconds or minutes, that the reagents are allowed to react at particularpositions of the channel is shown to the right of the main channel. Adetection window or zone is shown as a dashed box. FIG. 1B illustrates asituation where the flow rate is increased, e.g., to 1.67 times theoriginal flow rate. In doing so, the reagents are allowed to react for ashorter amount of time before they move past the detection zone, becausethey are moving faster. Conversely, FIG. 1C illustrates a situationwhere the reagents are moving more slowly, thus allowing them more timeto react within the channel prior to moving through the detection zone.

In accordance with the present invention, flow rate of the reactionmixture is typically varied in order to monitor the extent of a givenreaction at different reaction times. In particularly preferred aspects,this variation of flow rate is done in real time, or “on-the-fly,”meaning that the operation of the system is not stopped during the flowrate variation. Also, although the flow rate may be varied in steps,e.g., sharp increases or decreases in flow rate, in preferred aspects,the present invention is directed to a more gradual ramping up or downof the flow rate. In similarly preferred aspects, the flow rates arevaried in accordance with pre-designated or programmed instructions soas to produce a desired flow-rate profile of reaction mixture throughthe main flow channel.

In an alternate aspect, different time points of a given reaction aremonitored by monitoring different positions within the flow channel.This may be accomplished using multiple detectors to detection reactionresults, or by using a single detector that is movable among thedifferent points along the channel.

II. Systems

As noted above, the present invention typically utilizes a flow channelto contain the reaction mixtures of interest. The various reagents thatmake up the reaction mixture are introduced into the main reactionchannel via one or more connecting channels. In a simple embodiment, theflow channel comprises a single flow conduit, i.e., a tube, capillary orother flow channel that is separately connected to the reagent sourcesso as to allow concurrent introduction of the various reagents into themain flow channel such that the reagents mix within or just prior totheir introduction into the main flow channel.

In particularly preferred aspects, the flow channels and connectingchannels are incorporated into an integrated microfluidic device. Asused herein, the term “microfluidic” typically refers to a fluidpassageway or conduit that has at least one microscale cross-sectionaldimension, e.g., from about 0.1 to about 500 μm. Integrated microfluidicdevices typically comprise a network of microscale fluidic elements,e.g., conduits, chambers or the like, that are fluidly coupled to oneanother. While such systems may be embodied in an aggregation ofseparate elements, e.g., capillaries, chambers, etc., in preferredaspects, they are integrated into a single planar body structure.Typically, these planar body structures are fabricated from anaggregation of substrate layers having features fabricated on theirsurfaces such that the fluidic elements are defined at the interfaces ofthe substrate layers. In particular, a planar surface of one or more ofthe substrate layers is fabricated to define a series of grooves and ordepressions therein. A planar surface of a second substrate layer isthen overlaid and bonded to the surface of the first to seal the groovesor depressions to form the sealed channels or chambers of the device.

FIG. 2 schematically illustrates a device incorporating this layeredstructure. As shown, the device 10 includes a first planar substrate 12having an upper surface 14. A plurality of grooves and/or depressions 16are defined in the upper surface 14 of the first substrate 12. A secondsubstrate 18 having a lower planar surface 20 and an upper planarsurface 22 is then placed upon the upper surface 14 on the lowersubstrate 12 and the substrates are bonded together to seal thegrooves/depressions to define channels/chambers within an interiorportion of the overall body structure. The upper surface 22 of thesecond substrate 18 may include one or more wells or reservoirs 24 whichare configured to be in fluid communication with the channel networkdefined by channels 16 in the lower substrate 12. Although described interms of grooves or depressions fabricated into the surface of one ofthe substrate layers, it will be appreciated that grooves may beprovided in either or both of the opposing surfaces of the twosubstrates so as to define more complex channel networks. Also, althoughillustrated as a single interconnected channel network, it will beappreciated that a single integrated device may include severaldiscrete, e.g., completely separate, channel networks or individualchannels.

FIG. 3 is a schematic illustration of a microfluidic device channelnetwork that is particularly suited for the methods of the presentinvention. As shown, the device 300 includes a plurality of differentreservoirs 314–340, disposed in the body of the device 300. As shown,the reagent reservoirs 314–336 are grouped in pairs, e.g., reservoirs314:316, 318:320, 322:330, etc. Each of the reservoir pairs, e.g., pair314:316, is fluidly connected via associated channels, e.g., channels314 a and 316 a, respectively, to one of a plurality of analysischannels, e.g., channels 302–312. Each of the plurality of analysischannels is then coupled to one or more waste reservoirs, e.g.,reservoirs 338 and 340, via one or more waste channels, e.g., channels338 a and 340 a. A detection window or region 350 also exists over eachof the analysis channels 302–312, in order to observe the signalsemanating from those channels, e.g., fluorescent signals. Operation ofthe device shown in FIG. 3 in accordance with the present invention isdescribed in greater detail, below.

In carrying out the methods described herein, it is generally useful tocouple a fluid transport system to the flow channels described above.Typically, these flow systems are capable of directing the reactionmixtures through the main channel portions at varying flow rates. Asnoted above, in preferred aspects, the flow systems are capable of beingadjusted on-the-fly, either manually or in accordance with preprogrammedflow profiles that are input by a user. Typically, the flow systemscomprise a flow controller that is operably coupled to at least the mainflow channel in which the reaction monitoring is being carried out. By“operably coupled” is meant that the flow controller is coupled to thechannel in a way that permits the application of a flow driving force tothe contents of the flow channel so as to cause material movementthrough that channel.

As noted above, the controller instrument may include one or more of avariety of different flow system types. For example, in certainpreferred aspects, the flow controller includes a vacuum or pressuresource that is operably coupled to one or more channels of themicrofluidic device during operation to push or draw fluid through thechannels of the device. In alternative preferred aspects, the controllercomprises a source of electrical power that is operably coupled to thechannels of the device, e.g., via electrodes inserted into reservoirs atthe termini of channels, to move fluid or other materials through thechannels of the device via electrokinetic forces, i.e., electrophoresisand/or electroosmosis.

In the case of a vacuum or pressure source, this typically comprisesvacuum or pressure pump integrated into the instrument. Typically,commonly used pumps, such as syringe pumps, peristaltic pumps or thelike are preferred for use in these applications. In accordance with thepresent invention, these pumps also are capable of varying the amount ofpressure or vacuum supplied to the channels of the device, and therebyvary the flow rate of material through those channels, e.g., an increasein the pressure differential increases the flow rate, while a decreasein the pressure differential results in a decreased flow rate. Thevariation of the flow rate is generally carried out in accordance with auser input flow profile that is programmed either into the controllerunit or into a computer that is operably coupled to the controller. By“user input” is meant that a flow profile instruction is provided to thecontroller instrument trough a programming step, e.g., as firmware orsoftware that is input by the end-user or during the manufacturingprocess for the controller instrument or its associated computer.

The pressure or vacuum sources are typically provided operably coupledto one or more of the channels of the microfluidic device to controlflow therethrough. For example, with respect to the microfluidic deviceillustrated in FIG. 3, one or more pressure sources may be providedcoupled to ports 314–340 in order to provide a pressure differentialthrough the channels connected to those ports. Appropriate control ofthe pressures applied at the ends of the various channels permitscontrol of the relative flow of material through those channels.

In particularly preferred aspects, flow rate within interconnectedchannels is controlled at a single port to the channels, e.g., a commonwaste port. Specifically, again with reference to FIG. 3, a negativepressure or vacuum is applied at a terminal or waste reservoir or port,e.g., reservoir 338 and/or 340. This vacuum draws fluid through thevarious channels of the device, e.g., channels 302–312 toward the wastereservoirs. The various channels are configured such that fluid flowsthrough the channels at a predetermined rate relative to other channelsunder the applied vacuum. Typically, the configuration of these channelsinvolves providing the channels with a selected length or cross-sectionso as to dictate the relative flow rate through the channel. Suchchannel configuration is described in detail in, e.g., U.S. patentapplication Ser. No. 09/238,467, filed Jan. 28, 1999, which isincorporated herein by reference in its entirety for all purposes.

In the case of electrically controlled flow systems, varying flow rateswithin the channels of the device is typically accomplished by varyingthe electrokinetic force applied to the material in the channel. This istypically accomplished by increasing or decreasing the voltage dropacross the length of a particular channel through which material isbeing moved. Variation of applied voltages or current flow through achannel is also typically accomplished in accordance with a user inputflow profile. Connection of the channels to electrical power sources istypically accomplished by providing electrodes that are connected to thepower supply in direct contact with fluid within the reservoirs of thedevice, e.g., reservoirs 314–340 of FIG. 3.

The flow channel in which the particular reaction is being monitoredincludes a detection zone at which the results of the operation aredetected. The detection zone is marked by the presence of a detectionsystem that is positioned so as to be in sensory communication with thecontents of the flow channel at the detection zone. As used herein, thephrase “within sensory communication” refers to a detection system thatis positioned so as to monitor a particular detectable event within thedetection zone. These detectable events may be optical (e.g.,fluorescent signals, absorbance characteristics, colored signals, lightscattering signals, or other changes in optical characteristics, e.g.,refractive index, etc.), thermal, electrochemical (conductivity),chemical (chemical constituents, e.g., oxygen, etc.) or physical(viscosity, etc.) in nature.

In the case of optical signals, a detection system that is in sensorycommunication with a particular material is positioned so as to receivea sufficient quantity of the optical signal so as to detect itspresence. Typically, this is carried out by positioning an opticaldetection system adjacent to a transparent or translucent portion of theflow channel, e.g., the detection zone. An optical signal is thentransmitted through the transparent or translucent portion of the flowchannel and collected and detected by the detection system. In the caseof electrochemical or chemical detection, such systems often require thepresence of specific sensors in direct physical contact with thecontents of the flow channel, e.g., as monitoring electrodes, or thelike, in order to be in sensory communication with the contents of theflow channel. Optical signals and detection systems are most preferredin the present invention for their ease of use, and physical (althoughnot sensory) isolation from the contents of the flow channel. Althoughnot required, in preferred aspects, the detection system is integratedwith the flow controller system into a single instrument.

Detection may be carried out at a single point along the flow channel,e.g., using a point detector such as a laser based fluorescencedetector. Such detection is useful where a single data point is requiredfor determining analysis results. Alternatively, a wider area of theflow channel may be monitored for detecting the signal, e.g., using ascanning system that scans a larger portion of a channel, an imagingsystem such as a CCD or a linear laser based detector, where analysis ofresults requires observation of the signal within a given channel for alonger time period. In cases where multiple channels are being observed,either for multiple time points of a single reaction (as described withreference to FIG. 8) or among multiple different channels in whichdifferent reactions are being monitored, e.g., as shown in FIG. 3. Avariety of linear laser detectors are known in the art, including, e.g.,galvo-scanners, cylindrical lens detectors in combination with imagingsystems, i.e., that focus excitation light in a linear pattern. Inparticularly preferred aspects, the controller/detector function isprovided by a platform instrument available from, e.g., AgilentTechnologies, Inc. and/or Caliper Technologies Corp., Specifically, forintegrated microfluidic devices having sample sources incorporatedtherein, the device can be placed into a 2100 Bioanalyzer available fromAgilent Technologies, Inc., optionally outfitted with a vacuum port forpressure/vacuum control of at least one port of the device.Alternatively, an HTS system available from Caliper Technologies Corp.,under their Technology Access Program, permits the accession oflibraries of sample materials contained in standard storage systems,e.g., multiwell plates (see www.calipertech.com).

In addition, as noted above, another method of measuring different timepoints of a reaction involves moving the detector to different pointsalong the flow channel while otherwise maintaining flow through thechannel at a constant level. In particularly preferred aspects, the flowchannel is arranged in a serpentine or reciprocating pattern at thedetection zone, such that ensuing portions of the flow channel turn backand run parallel to the preceding portions of the channel. A detector isprovided that can simply scan or step from one portion of the channel toanother portion, etc., to detect the reaction results at that point. Anexample of a channel geometry for accomplishing this is shown in FIG. 8.Briefly, the flow channel 802 is connected to one or more reagentsources, e.g., via channels 804 and 806, and includes a serpentinedetection region 808. The detection window 810 spans the varioussegments of the channel in the serpentine region 808. The geometry ofthe serpentine region and/or the flow rate through the flow channel canbe varied to alter the time intervals between the detection points. Avariety of scanning detection systems can be employed as the detectionaspect of the system, including track mounted detectors, pivotingdetectors and galvoscanner detectors.

III. Flow Rate Variation

As noted above, the present invention involves varying the rate of flowof reactants along a flow channel in order to monitor time dependentreactions involving those reactants (e.g., between two reactants, orinvolving only a single reactant). Specifically, at least a firstreagent or reactant is flowed into the flow channel and a reaction isinitiated at a first point in the flow channel. In typical cases, thisinvolves the combination of the first reactant with at least a secondreactant at a first point in a flow channel. Once the reaction isinitiated, a “reaction mixture” is formed which includes the reagentsand/or any products that result from the reaction. The reaction mixtureis then continuously flowed along the channel past a detection zone. Asnoted herein, initiation of the reaction optionally involves combinationof the first reagent with one or more additional reagents, but can alsoinvolve exposure of the first reagent to some external stimuli, e.g.,light or heat energy. In continuous flow assays, the initiation of thereaction, whether based upon addition of reagents or otherwise takesplace at a first point in the flow channel and is carried outcontinuously on the flowing stream of reagents. The detection of theresults of the reaction is then carried out at a second point in theflow channel. By varying the flow rate of the reactants between thefirst and second points, one can vary the reaction time beforedetection. Similarly, by varying the location of the second pointrelative to the first point, one can detect reactions that have varyingincubation/reaction times. Relatedly, one can simultaneously detect atmultiple different points along the flow channel that differ in theirlocations relative to the first point, in order to simultaneously obtainresults for the numerous reaction times. This is discussed in greaterdetail below.

By way of example and with reference to FIG. 3 showing a microfluidicdevice 300 for performing a plurality, e.g., 6 time dependent reactions,flow rate is varied along the flow channels 302–312 in order to monitorthe time dependent interaction of reactants. In particular, withreference to one flow channel 302, a first reactant is deposited intoone reservoir that is coupled to an upstream portion (from the detectionzone) of the flow channel 302, e.g., reservoir 322. A second reagent isdeposited into a second reservoir, e.g., reservoir 330, that also iscoupled to an upstream portion of the flow channel 302.

The reagents are each flowed into flow channel 302 from reservoirs 322and 330 via channels 322 a and 330 a, respectively, by either applying apositive pressure to reservoirs 322 and 330 or by applying a negativepressure to one of waste reservoirs 338 or 340. The reagents flow intoflow channel 302 and are mixed at intersection 342, whereupon the mixedreagents continue along flow channel 302 past a detection zone 350. Thedetected reagents then continue through the flow channel 302 into wastechannel 340a toward waste reservoir 340. The same operation occurs inparallel in each of flow channels 304–312, transporting reagents fromthe reservoirs associated with these flow channels. As shown, the flowchannels and connecting channels that couple the flow channels to theirassociated reservoirs are configured so as to provide similar if notidentical flow reagents into and through each of flow channels 302–312.

In the case of a single vacuum source applied at waste reservoir 340,variation of the flow rate along the flow channels 302–312 isaccomplished-by increasing or decreasing the level of vacuum applied atthe waste reservoir 340. An optional standard reservoir 338 is alsoshown. Typically, a dye standard is placed into this well in order toallow auto-focusing of the optical detection system upon channel 338 aat detection window 350. The change in vacuum results in a change in theamount of time that the reagents are mixed together prior to detection,e.g., changing the amount of time the reagents are present betweenintersection 342 and detection zone 350. In the case of the device shownin FIG. 3, up to 6 different reagent mixtures are tested in accordanceto the methods described herein. These different reagent mixtures may becombinations of different reagents, or the same reagents at differentconcentrations.

In some cases, it may be desirable to be able to produce a range of flowrates that is not easily achieved using a single flow channel,vacuum/pressure source combination. Specifically, in a single flowchannel, one can generally achieve a particular range of flow rates byvarying the motive force applied to the material in that channel due tothe responsive range of the controller when combined with a givenchannel. However, in a system employing multiple flow channels, e.g.,flow channels 302–312 in FIG. 3, one can further vary flow rates betweenchannels by varying the flow resistance of the different channels, e.g.,by varying their lengths and/or cross-sectional dimensions. Thus, whereall the channels of varied resistance are subjected to the same appliedmotive force, e.g., a vacuum source applied at a single waste reservoircoupled to each channel, the flow rates within the different channelswill be different. The flow rate within each of the channels is thenfurther varied by varying the applied motive force, e.g., pressure,vacuum, electric field. One can configure the different channels suchthat each will operate in a predetermined flow-rate range so as toprovide a much wider range of flow rates, and thus reaction times, thanis achieved using a single channel. Such configuration methods aredescribed in detail in U.S. patent application Ser. No. 09/238,467,previously incorporated herein by reference.

One can also vary flow resistance within the flow channel by varying theviscosity of the fluid that is being flowed through the channel.Typically, viscosity adjustments to a particular fluid involve theaddition of higher viscosity additives to the fluid, or its dilutionwith a lower viscosity fluid. Typically, viscosity raising additivesinclude, e.g., polymer solutions, e.g., PEG, polyacrylamide, cellulosicpolymers, ficoll, polyalcohols, i.e., polyvinylalcohols, glycerol,polypropanol, and the like.

IV. Applications

The methods, devices and systems of the invention are generally used inmonitoring reactions, and particularly the kinetic characteristics ofthose reactions. Generally, reactions include chemical reactions, aswell as biochemical and biological reactions between at least twodifferent reagents. Such reactions include simple chemical reactions,e.g., between two or more chemical reagents, to more complex biochemicalreactions, e.g., enzyme reactions, binding reactions, signalingreactions, complex cellular reactions, etc.

In preferred aspects, the methods and systems described herein are usedin the characterization and/or monitoring of biochemical or biologicalreactions. Typically such reactions are of particular interest in thebiological, biotechnical, diagnostic and/or pharmaceutical fields.Examples of biochemical reactions that are monitored in accordance withthe present invention include simple enzyme reactions, binding reactionsbetween specific binding pairs, e.g., ligand-receptor binding, nucleicacid binding reactions, protein/nucleic acid binding reactions,protein-protein interactions, and the like, as well as complex cellularreactions, e.g., cellular activation or signaling cascade reactions,cellular viability reactions, or the like.

In the case of simple chemical and/or biochemical reactions, theconstituent reagents for the reaction are introduced into and mixedwithin the flow channel. The reaction mixture is then transported alongthe flow channel through the detection zone, at which the reactionresults are detected. Variation of the flow rate provides a variation inthe reaction time after which results are detected, thereby providing areaction time course. A reaction time course obtained in this manner mayuse reaction results at a plurality of discrete reaction time points,e.g., based upon a stepping of reaction rates, or may use results from aconstantly changing reaction time, e.g., based upon a ramping flow rate.In particular, where specific predetermined reaction times are desiredto be monitored, a flow rate is held for a period of time, e.g., fromseveral seconds to several minutes, so as to provide a steady statelevel of reaction product that is detected. The flow rate of thereagents is then stepped to a different level and held for another timeperiod until a steady state is achieved. The reaction results for eachstep are correlated with the amount of reaction time for each step. Fromthis data, the reaction kinetics are calculated.

Briefly, the relevant experimental parameters affecting the reactiontimes (t) in the flow channel system described herein, are the appliedpressure differential (P), the hydrodynamic resistance of the channel(R), the reaction channel length (L) between the point of initiation ofthe reaction and the detection point, and the cross-sectional area ofthe channel. In the case where all of the reaction constituents movewith the bulk fluid velocity, the reaction time for a given pressuregradient P can be expressed as:t=LRA/PThus, understanding all of the applied parameters of the system, e.g.,L, R, A and P, one can determine the reaction time for results obtainedat any given pressure. Typically, the system parameters are eithermeasurable or are known from the specifications of the system asproduced. The various time-based kinetic parameters of the reaction canthen be calculated in accordance with well known procedures (See, e.g.,Segel, Biochemical Calculations, 2nd Ed (John Wiley and Sons 1976),incorporated herein by reference in its entirety for all purposes.

In particularly preferred aspects, at least one of the reagents that arebeing mixed are in the form of whole cell systems. In particular, suchsystems monitor the interaction of a cell system with another externalreagent that is mixed with the cells. This is typically accomplished bymonitoring the cell's response to the other reagent. A large number ofdifferent cellular responses may be monitored, depending upon theinformation that is desired. For example, a change in the cell'stransport functions, e.g., ion flux, the cell's ability to express geneproducts, viability or the like are readily monitored as importantbiological reactions. A variety of such important cell-based reactionsare described in co-pending U.S. patent application Ser. No. 09/104,519,filed Jun. 25, 1998, which is incorporated herein by reference in itsentirety for all purposes. In accordance with the present invention, thetime course of a cellular response to a particular chemical stimulus,e.g., reagent, is monitored over time using the methods describedherein. Specifically, a cell suspension is flowed into the flow channeland combined with a particular reagent and the effect of that reagentupon the cells is monitored at the detection zone. The time course ofthat particular reaction is then monitored by altering the flow rate ofthe mixture.

The present invention is further illustrated with reference to thefollowing non-limiting examples.

EXAMPLE 1 Kinetic Analysis

The above-described methods were used to monitor the time dependentresponse of cell systems to a variety of different chemical stimuli. Thedevice used for analysis was that shown in FIG. 3, and where useful forunderstanding, the same reference numerals are used.

For the following experiments, two different model systems were used. Inthe first model system, a non-adherent THP-1 human monocytic leukemiacell line, expressing an endogenous P2 purinergic receptor was exposedto UTP as the ligand, and calcium flux within the cells was monitored.In the second system, an adherent CHO-M1 chinese hamster ovary cell lineexpressing a transfected M1 muscarinic receptor was exposed to carbacholas the ligand, and again, calcium flux was monitored.

In both systems, the cells were loaded with the calcium-sensitivefluorescent indicator dye Fluo-4 (Molecular Probes). The cells were alsostained with a fluorescent nucleic acid stain Syto 62 (Molecular Probes)that ubiquitously stains all cells. The cells were washed andresuspended in isotonic and isopynic buffer (Hank's Balanced SaltSolution (HBSS) pH 7.4, 18% (v/v) Optiprep, 20 mM HEPES, 0.135% BSA,referred to herein as “Buffer A”). The ligand was prepared in six doses(half-log dilutions) in Buffer A.

The channels of a microfluidic device having the channel structure shownin FIG. 3 were flushed with Buffer A. The cell suspension and ligandwere added to their respective wells in the microfluidic device, e.g.,cell suspensions in wells 314, 318, 322, 324, 326 and 328, and ligandsolution in wells 316, 320, 330, 332, 334 and 336. A fluoresceinstandard was added to the dye well 338 in order to facilitateauto-focusing of the optical detection system. The device was insertedin a modified Agilent 2100 Bioanalyzer that was fitted with a vacuumport that interfaced with waste well 340 of the device. The opticaldetection system utilized a blue LED as the excitation source and thestandard Bioanalyzer optics.

After the run was started, the detector autofocused on the dye channel(338 a) and moved sequentially across each of the analysis channels,collecting data from each channel for the entire course of the vacuumvariation before moving to the next channel.

For each channel, the calcium signal was normalized with Syto 62 dye.The mean normalized calcium signal for each pressure step wascalculated. The normalized calcium signal was then plotted against thepressure step/incubation time, to generate the kinetic curve of thereaction. FIG. 5 illustrates this curve for the response of the P2ureceptor to UTP. FIG. 7 illustrates the same kinetic curve for theresponse of the M1 muscarinic receptor to Carbachol. The peak responsevalues from the kinetic curves were then plotted against the respectiveligand concentration (log) to give the dose response curve for eachsystem, which are shown in FIGS. 4 and 6, for the P2u receptor and M1muscarinic receptor, respectively.

Unless otherwise specifically noted, all concentration values providedherein refer to the concentration of a given component as that componentwas added to a mixture or solution independent of any conversion,dissociation, reaction of that component to alter the component ortransform that component into one or more different species once addedto the mixture or solution.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. Although the present invention has beendescribed in some detail by way of illustration and example for purposesof clarity and understanding, it will be apparent that certain changesand modifications may be practiced within the scope of the appendedclaims.

1. A method of monitoring a time dependent reaction, comprising: continuously flowing at least a first reagent through a first flow channel; initiating a reaction with the first reagent at a first point in the first flow channel; detecting a result of the reaction at a second point in the first flow channel; varying an amount of time between the initiation of the reaction and the detection with respect to the first reagent flowing through the first flow channel by varying a rate at which the first reagent flows through the first flow channel from the first point to the second point; and performing at least one detection at each of multiple flow rates, thereby monitoring a time dependent reaction.
 2. The method of claim 1, wherein the step of initiating the reaction of the first reagent comprises mixing a second reagent with the first reagent to form a first reaction mixture.
 3. The method of claim 2, wherein at least one of the first and second reagents comprises an enzyme.
 4. The method of claim 2, wherein at least one of the first and second reagents comprises at least one member of a specific binding pair.
 5. The method of claim 4, wherein the specific binding pair is from a ligand-receptor pair, complementary nucleic acids, a nucleic acid-nucleic acid binding protein pair, or an antibody-antigen pair.
 6. The method of claim 1, wherein the reaction produces an optically detectable signal.
 7. The method of claim 6, wherein the optically detectable signal comprises a fluorescent signal.
 8. The method of claim 7, wherein the fluorescent signal comprises an increase or decrease in a level of fluorescence in the first flow channel.
 9. The method of claim 7, wherein the fluorescent signal comprises a change in an amount of depolarized fluorescence within the first flow channel.
 10. The method of claim 2, wherein the first flow channel and at least one of a source of the first reagent and a source of the second reagent are disposed in an integrated microfluidic body structure.
 11. The method of claim 1, wherein the step of varying the flow rate comprises varying an applied pressure differential along the length of the first flow channel.
 12. The method of claim 11, wherein the step of varying an applied pressure differential along a length of the first flow channel comprises varying a vacuum applied at one end of the first flow channel.
 13. The method of claim 11, wherein the step of varying an applied pressure differential along a length of the first flow channel comprises varying a positive pressure applied at one end of the first flow channel.
 14. The method of claim 2, further comprising: providing at least a second flow channel; introducing the first and second reagents into the second flow channel whereupon the first and second reagents mix to form a second reaction mixture, at least one of the first or second reagent being present in the second reaction mixture at a concentration different from its concentration in the first reaction mixture; varying a flow rate of the second reaction mixture along the second flow channel; and monitoring a result of an interaction between the first and second reagents.
 15. The method of claim 2, further comprising: providing at least a second flow channel; introducing third and fourth reagents into the second flow channel whereupon the third and fourth reagents mix to form a second reaction mixture, at least one of the third and fourth reagents being different from the first and second reagents; varying a flow rate of the second reaction mixture along the second flow channel; and monitoring a result of an interaction between the third and fourth reagents.
 16. The method of claim 2, further comprising: providing at least a second flow channel, the second flow channel having a flow resistance that is different from a flow resistance of the first flow channel; introducing the first and second reagents into the second flow channel, whereupon the first and second reagents mix to form a second reaction mixture; varying a flow rate of the second reaction mixture along the second flow channel; and monitoring a result of an interaction between the first and second reagents in the second reaction mixture.
 17. The method of claim 16, wherein the steps of varying the flow rate of the first reaction mixture along the first flow channel and the second reaction mixture along the second flow channel comprises applying a single pressure differential across a length of the first and second flow channels, the different flow resistance of the second flow channel from the first flow channel producing a different flow rate of the second reaction mixture through the second flow channel than for the first reaction mixture through the first flow channel.
 18. The method of claim 17, wherein the first and second flow channels are fluidly connected to a common port, and the step of varying the flow rate of the first reaction mixture along the first flow channel and the second reaction mixture along the second flow channel comprises applying a positive pressure or vacuum to the common port to move the first and second reaction mixtures through the first and second flow channels, respectively. 